Scanning Probe Microscope Tips
Sophisticated protein molecules are like people in that the behaviors of individuals of the same population are different. While studying these molecules with traditional methods such as X-ray diffraction resembles the “social science of molecules,” providing a statistically averaged property of an ensemble of molecules at static states, the heterogeneity, dynamics, and distribution of the properties among individual molecules is masked. Recent advances in single molecule methods has allowed study of the structure-function relationship of individual molecules, and exploration of heterogeneity among different molecules within a population, as well as observation of action of individual molecules in real time. The research in this new field, “single molecule biochemistry,” promises to provide fundamentally new information of biological processes for a better understanding of cellular function. The progress in this field has heavily relied on the development of tools for detection and manipulation of single molecules.
AFM has become a powerful tool for biological research at nanoscale [Binnig, G., Quate, C. F. & Gerber, C. Atomic force microscope. Phys. Rev. Lett. 56, 930 (1986); Science Citation Index. Search keyword: AFM or atomic force microscopy]. AFM uses a cantilever spring with a sharp tip to sense the repulsive and attractive forces between the tip and a sample surface. Commercial AFM can measure the forces as low as 10 piconewtons needed to rapidly rupture a single hydrogen bond [Takano, H., Kenseth, J. R., Wong, S. S., O'Brien, J. C. & Porter, M. D. Chemical and biochemical analysis using scanning force microscopy. Chem. Rev. 99, 2845 (1999); Poggi, M. A., Bottomley, L. A. & Lillehei, P. T. Scanning probe microscopy. Anal. Chem. 74, 2851 (2002); Hoh, J. H., Cleveland, J. P., Prater, C. B., Revel, J. P. & Hansma, P. K. Quantized adhesion detected with the atomic force microscope. J. Am. Chem. Soc. 114, 4917 (1992); Zlatanova, J., Lindsay, S. M. & Leuba, S. H. Single molecule force spectroscopy in biology using the atomic force microscope. Prog. Biophys. Mol. Biol. 74, 37 (2000)]. While scanning over the surface, the data of tip-sample interaction provides images of the surface with a high spatial resolution. Both the high force and spatial resolutions render AFM feasible to probe non-covalent inter- and intramolecular interactions at molecular level. To this end, the surface of AFM tips, mostly made of silicon or silicon nitride, have been chemically or biochemically modified for probing specific interactions between the molecules attached to the tip and their partners immobilized on surfaces [Willemsen, O. H., Snel, M. M. E., Cambi, A., Greve, J., De Grooth, B. G. & Figdor, C. G. Biomolecular interactions measured by atomic force microscopy. Biophys. J. 79, 3267 (2000); Tamayo, J., Humphris, A. D. L., Owen, R. J. & Miles, M. J. High-Q dynamic force microscopy in liquid and its application to living cells. Biophys. J. 81, 526 (2001); Wang, T., Arakawa, H. & Ikai, A. Force measurement and inhibitor binding assay of monomer and engineered dimer of bovine carbonic anhydrase B. Biochem. Biophys. Res. Commun. 285, 9 (2001); Nakagawa, T., Ogawa, K., Kurumizawa, T. & Ozaki, S. Discriminating Molecular Length of Chemically Adsorbed Molecules Using an Atomic Force Microscope Having a Tip Covered with Sensor Molecules (an Atomic Force Microscope Having Chemical Sensing Function). Jpn. J. Appl. Phys. Part 2—Lett. 32, L294 (1993); Frisbie, C. D., Rozsnyai, L. F., Noy, A., Wrighton, M. S. & Lieber, C. M. Functional-Group Imaging by Chemical Force Microscopy. Science 265, 2071 (1994); Noy, A., Vezenov, D. V. & Lieber, C. M. Chemical force microscopy. Annu. Rev. Mater. Sci. 27, 381 (1997); Hugel, T. & Seitz, M. The study of molecular interactions by AFM force spectroscopy. Macromol. Rapid Commun. 22, 989 (2001); Janshoff, A., Neitzert, M., Oberdorfer, Y. & Fuchs, H. Force spectroscopy of molecular systems—Single molecule spectroscopy of polymers and biomolecules. Angew. Chem.-Int. Edit. 39, 3213 (2000); Tromas, C. & Garcia, R. in Host-Guest Chemistry 115 (2002)], such as biotin-streptavidin/aviding [Schonherr, H., Beulen, M. W. J., Bugler, J., Huskens, J., van Veggel, F., Reinhoudt, D. N. & Vancso, G. J. Individual supramolecular host-guest interactions studied by dynamic single molecule force spectroscopy. J. Am. Chem. Soc. 122, 4963 (2000); Lee, G. U., Kidwell, D. A. & Colton, R. J. Sensing Discrete Streptavidin Biotin Interactions with Atomic-Force Microscopy. Langmuir 10, 354 (1994)], antigen-antibody [Moy, V. T., Florin, E. L. & Gaub, H. E. Intermolecular Forces and Energies between Ligands and Receptors. Science 266, 257 (1994); Hinterdorfer, P., Baumgartner, W., Gruber, H. J., Schilcher, K. & Schindler, H. Detection and localization of individual antibody-antigen recognition events by atomic force microscopy. Proc. Natl. Acad. Sci. U.S.A. 93, 3477 (1996); Hinterdorfer, P., Gruber, H. J., Kienberger, F., Kada, G., Riener, C., Borken, C. & Schindler, H. Surface attachment of ligands and receptors for molecular recognition force microscopy. Colloid Surf. B-Biointerfaces 23, 115 (2002)], and complementary strands of DNA [Raab, A., Han, W. H., Badt, D., Smith-Gill, S. J., Lindsay, S. M., Schindler, H. & Hinterdorfer, P. Antibody recognition imaging by force microscopy. Nat. Biotechnol. 17, 902 (1999); Wielert-Badt, S., Hinterdorfer, P., Gruber, H. J., Lin, J. T., Badt, D., Wimmer, B., Schindler, H. & Kinne, R. K. H. Single molecule recognition of protein binding epitopes in brush border membranes by force microscopy. Biophys. J. 82, 2767 (2002); Lee, G. U., Chrisey, L. A. & Colton, R. J. Direct Measurement of the Forces between Complementary Strands of DNA. Science 266, 771 (1994); Boland, T. & Ratner, B. D. Direct Measurement of Hydrogen-Bonding in DNA Nucleotide Bases by Atomic-Force Microscopy. Proc. Natl. Acad. Sci. U.S.A. 92, 5297 (1995); Noy, A., Vezenov, D. V., Kayyem, J. F., Meade, T. J. & Lieber, C. M. Stretching and breaking duplex DNA by chemical force microscopy. Chem. Biol. 4, 519 (1997); Clausen-Schaumann, H., Rief, M. & Gaub, H. E. Sequence dependent mechanics of single DNA molecules. Biophys. J. 76, A151 (1999)]. Chemically modified AFM tips have also been used to harvest strands of genomic DNA at a specific region of a chromosome, and the DNA amplified by PCR [Clausen-Schaumann, H., Rief, M., Tolksdorf, C. & Gaub, H. E. Mechanical stability of single DNA molecules. Biophys. J. 78, 1997 (2000); Schumakovitch, I., Grange, W., Strunz, T., Bertoncini, P., Guntherodt, H. J. & Hegner, M. Temperature dependence of unbinding forces between complementary DNA strands. Biophys. J. 82, 517 (2002)]. By tethering a polymer strand both to an AFM tip and a solid surface, the polymer can be pulled by the AFM while the force-extension curves are recorded, which provide insight into the structural and mechanical properties of the molecule, including rupture force of a single covalent bond [Xu, X. M. & Ikai, A. Retrieval and amplification of single-copy genomic DNA from a nanometer region of chromosomes: A new and potential application of atomic force microscopy in genomic research. Biochem. Biophys. Res. Commun. 248, 744 (1998)], and the intramolecular forces associated with protein folding [Hoh, J. H., Cleveland, J. P., Prater, C. B., Revel, J. P. & Hansma, P. K. Quantized adhesion detected with the atomic force microscope. J. Am. Chem. Soc. 114, 4917 (1992); Grandbois, M., Beyer, M., Rief, M., Clausen-Schaumann, H. & Gaub, H. E. How strong is a covalent bond? Science 283, 1727 (1999); Rief, M., Gautel, M., Oesterhelt, F., Fernandez, J. M. & Gaub, H. E. Reversible unfolding of individual titin immunoglobulin domains by AFM. Science 276, 1109 (1997)] and elasticity and conformational transitions in such as polysachharides [Oberdorfer, Y., Fuchs, H. & Janshoff, A. Conformational analysis of native fibronectin by means of force spectroscopy. Langmuir 16, 9955 (2000); Muller, D. J. & Engel, A. in Atomic Force Microscopy in Cell Biology 257 (2002)] and DNA [Noy, A., Vezenov, D. V., Kayyem, J. F., Meade, T. J. & Lieber, C. M. Stretching and breaking duplex DNA by chemical force microscopy. Chem. Biol. 4, 519 (1997); Clausen-Schaumann, H., Rief, M. & Gaub, H. E. Sequence dependent mechanics of single DNA molecules. Biophys. J. 76, A151 (1999); Li, H. B., Rief, M., Oesterhelt, F., Gaub, H. E., Zhang, X. & Shen, J. C. Single-molecule force spectroscopy on polysaccharides by AFM—nanomechanical fingerprint of alpha-(1,4)-linked polysaccharides. Chem. Phys. Lett. 305, 197 (1999); Marszalek, P. E., Li, H. B., Oberhauser, A. F. & Fernandez, J. M. Chair-boat transitions in single polysaccharide molecules observed with force-ramp AFM. Proc. Natl. Acad. Sci. U.S.A. 99, 4278 (2002); Fisher, T. E., Marszalek, P. E. & Fernandez, J. M. Stretching single molecules into novel conformations using the atomic force microscope. Nat. Struct. Biol. 7, 719 (2000); Bustamante, C., Macosko, J. C. & Wuite, G. J. L. Grabbing the cat by the tail: Manipulating molecules one by one. Nat. Rev. Mol. Cell Biol. 1, 130 (2000)].
Most of the chemically and biochemically modified AFM tips were derived from a layer of small molecules either on a gold-coated or directly on a Si/Si3N4 tip. When these tips are in contact with a sample surface, multiple to more than tens of molecules on the tip can participate semi-simultaneously in the binding or unbinding events. With such systems, the mean value of single-molecule interaction force can be derived by statistical treatment of data obtained from many repeated measurements of the pull-off force [Takano, H., Kenseth, J. R., Wong, S. S., O'Brien, J. C. & Porter, M. D. Chemical and biochemical analysis using scanning force microscopy. Chem. Rev. 99, 2845 (1999); Poggi, M. A., Bottomley, L. A. & Lillehei, P. T. Scanning probe microscopy. Anal. Chem. 74, 2851 (2002); Williams, M. C. & Rouzina, I. Force spectroscopy of single DNA and RNA molecules. Curr. Opin. Struct. Biol. 12, 330 (2002); Johnson, K. L., Kendall, K. & Roberts, A. D. Proc. R. Soc. London, Ser. A 324, 301 (1971); Stevens, F., Lo, Y. S., Harris, J. M. & Beebe, T. P. Computer modeling of atomic force microscopy force measurements: Comparisons of Poisson, histogram, and continuum methods. Langmuir 15, 207 (1999)]. However, this value is an average over an ensemble of multiple interaction sites. The ultimate goal of AFM is to address individual molecule at a specific site, especially if the molecule has multiple binding sites [Zlatanova, J., Lindsay, S. M. & Leuba, S. H. Single molecule force spectroscopy in biology using the atomic force microscope. Prog. Biophys. Mol. Biol. 74, 37 (2000)].
Most measurements of intra-molecular forces of single molecules including proteins and polysaccharides start with “fishing” a molecule with a “sticky” AFM tip [Hoh, J. H., Cleveland, J. P., Prater, C. B., Revel, J. P. & Hansma, P. K. Quantized adhesion detected with the atomic force microscope. J. Am. Chem. Soc. 114, 4917 (1992); Noy, A., Vezenov, D. V. & Lieber, C. M. Chemical force microscopy. Annu. Rev. Mater. Sci. 27, 381 (1997); Hugel, T. & Seitz, M. The study of molecular interactions by AFM force spectroscopy. Macromol. Rapid Commun. 22, 989 (2001); Li, H. B., Rief, M., Oesterhelt, F., Gaub, H. E., Zhang, X. & Shen, J. C. Single-molecule force spectroscopy on polysaccharides by AFM—nanomechanical fingerprint of alpha-(1,4)-linked polysaccharides. Chem. Phys. Lett. 305, 197 (1999); Marszalek, P. E., Li, H. B., Oberhauser, A. F. & Fernandez, J. M. Chair-boat transitions in single polysaccharide molecules observed with force-ramp AFM. Proc. Natl. Acad. Sci. U.S.A. 99, 4278 (2002); Fisher, T. E., Marszalek, P. E. & Fernandez, J. M. Stretching single molecules into novel conformations using the atomic force microscope. Nat. Struct. Biol. 7, 719 (2000)]. (Reference FIG. 9 for “fishing.”) The molecules are immobilized on a solid surface, and the tip is brought in contact with the molecules to establish a strong binding before the tip retracts away from the surface. The probability of “fishing” one or more molecules depends on the density of the molecules on the surface and the nature of the interaction including binding strength and number of binding sites on the tip and the molecule. Also, multiple bindings can occur randomly if the molecule possesses multiple binding sites. To minimize the attachment of multiple molecules, the so-called “fly-fishing mode” [Lo, Y. S., Huefner, N. D., Chan, W. S., Stevens, F., Harris, J. M. & Beebe, T. P. Specific interactions between biotin and avidin studied by atomic force microscopy using the Poisson statistical analysis method. Langmuir 15, 1373 (1999); Rief, M., Oesterhelt, F., Heymann, B. & Gaub, H. E. Single molecule force spectroscopy on polysaccharides by atomic force microscopy. Science 275, 1295 (1997)] has been used, in which the tip approaches the surface step by step, retracting partly after each approach until a binding event is observed in the force-extension curve upon pulling back. The presence of only one molecule between the tip and the surface is indicated by the characteristic conformational transitions of the molecule.
Few Single Molecule AFM Tips (SMAT) designed for studying intermolecular interactions have been reported. Hinterdorfer et al were the first to demonstrate the preparation of SMATs containing a polyethylene glycol (PEG) linker tethering with a polyclonal anti-HAS antibody molecule for interacting with an immobilized HSA antigen molecule [Moy, V. T., Florin, E. L. & Gaub, H. E. Intermolecular Forces and Energies between Ligands and Receptors. Science 266, 257 (1994)]. They measured the unbind force of the single antibody-antigen pair, and discovered that both binding sites of the antibody could bind simultaneously and independently with the same probability. In addition, they demonstrated the mapping of binding probability over the surface of the antigen molecule with a lateral resolution of 1.5 nm. Notably, the antibody molecules in the SMATs did not deteriorate after measuring thousands of force-extension curves and storage in buffer for more than two months [Moy, V. T., Florin, E. L. & Gaub, H. E. Intermolecular Forces and Energies between Ligands and Receptors. Science 266, 257 (1994)]. These SMATs were prepared by solution deposition of the PEG linker molecues to the tip using a condition leading to a surface density of about one molecule per 50 nm2 (about the area of an AFM tip apex). However, the precise number and location of the molecules at a given tip apex cannot be controlled by this method. The PEG molecules were flexible and long (8 nm) for overcoming mis-orientation, steric hindrance, and conformational changes for efficient antibody-antigen binding [Moy, V. T., Florin, E. L. & Gaub, H. E. Intermolecular Forces and Energies between Ligands and Receptors. Science 266, 257 (1994)]. The antibody at various locations at the tip apex may reach the antigen. However, the advantage of long and flexible PEG linker is limited for molecules anchored slightly away from the tip apex. In addition, the non-specific interactions between the bulk tip and the sample varies with the different locations of the molecules. Therefore, the reproducibility of the results obtained with different “SMATs” prepared by the above method is questionable.
Recently, Lieber and co-workers demonstrated the fabrication of SMATs on carbon nanotube modified tips [Sekiguchi, H., Arakawa, H., Okajima, T. & Ikai, A. Non-destructive force measurement in liquid using atomic force microscope. Appl. Surf. Sci. 188, 489 (2002); Dai, H. J., Hafner, J. H., Rinzler, A. G., Colbert, D. T. & Smalley, R. E. Nanotubes as nanoprobes in scanning probe microscopy. Nature 384, 147 (1996); Hafner, J. H., Cheung, C. L. & Lieber, C. M. Direct growth of single-walled carbon nanotube scanning probe microscopy tips. J. Am. Chem. Soc. 121, 9750 (1999); Hafner, J. H., Cheung, C. L. & Lieber, C. M. Growth of nanotubes for probe microscopy tips. Nature 398, 761 (1999)]. Single wall carbon nanotubes (SWNTs) have a small diameter (0.7–5 nm), and are the stiffest material, rendering SWNT-modified AFM tips ideal for high resolution imaging [Hafner, J. H., Cheung, C. L. & Lieber, C. M. Growth of nanotubes for probe microscopy tips. Nature 398, 761 (1999); Hafner, J. H., Cheung, C. L., Woolley, A. T. & Lieber, C. M. Structural and functional imaging with carbon nanotube AFM probes. Prog. Biophys. Mol. Biol. 77, 73 (2001)]. Carboxylic acids groups were generated by electrochemical etching at the tip terminal [Schnitzler, G. R., Cheung, C. L., Hafner, J. H., Saurin, A. J., Kingston, R. E. & Lieber, C. M. Direct imaging of human SWI/SNF-remodeled mono- and polynucleosomes by atomic force microscopy employing carbon nanotube tips. Mol. Cell. Biol. 21, 8504 (2001); Snow, E. S., Campbell, P. M. & Novak, J. P. Atomic force microscopy using single-wall C nanotube probes. J. Vac. Sci. Technol. B 20, 822 (2002); Wong, S. S., Woolley, A. T., Joselevich, E., Cheung, C. L. & Lieber, C. M. Covalently-functionalized single-walled carbon nanotube probe tips for chemical force microscopy. J. Am. Chem. Soc. 120, 8557 (1998); Hiura, H., Ebbesen, T. W. & Tanigaki, K. Opening and Purification of Carbon Nanotubes in High Yields. Adv. Mater. 7, 275 (1995)] and were used to attach biotin molecules [Hiura, H., Ebbesen, T. W. & Tanigaki, K. Opening and Purification of Carbon Nanotubes in High Yields. Adv. Mater. 7, 275 (1995)]. It is significant that the COOH groups thus generated are confined to the small tip end that is away from the bulk tip surface. However, the number of these groups varies with individual tips, and they may bond more than one biotin molecule, although single molecule tips can be identified by measurement of the unbinding force of the biotin-streptavidin complexes [Wong, S. S., Joselevich, E., Woolley, A. T., Cheung, C. L. & Lieber, C. M. Covalently functionalized nanotubes as nanometre-sized probes in chemistry and biology. Nature 394, 52 (1998); Sinnott, S. B. Chemical functionalization of carbon nanotubes. J. Nanosci. Nanotechnol. 2, 113 (2002)]. In addition, there exist non-specific interactions between the hydrophobic surface of carbon nanotubes and biomolecules, e.g. proteins, in many biological systems. Furthermore, nanotube tips are still relatively difficult to prepare, requiring chemical vapor deposition and transmission electron microscopy facilities [Dai, H. J., Hafner, J. H., Rinzler, A. G., Colbert, D. T. & Smalley, R. E. Nanotubes as nanoprobes in scanning probe microscopy. Nature 384, 147 (1996); Hafner, J. H., Cheung, C. L. & Lieber, C. M. Growth of nanotubes for probe microscopy tips. Nature 398, 761 (1999); Cheung, C. L., Hafner, J. H. & Lieber, C. M. Carbon nanotube atomic force microscopy tips: Direct growth by chemical vapor deposition and application to high-resolution imaging. Proc. Natl. Acad. Sci. U.S.A. 97, 3809 (2000); Wong, S. S., Joselevich, E., Woolley, A. T., Cheung, C. L. & Lieber, C. M. Covalently functionalized nanotubes as nanometre-sized probes in chemistry and biology. Nature 394, 52 (1998); Sinnott, S. B. Chemical functionalization of carbon nanotubes. J. Nanosci. Nanotechnol. 2, 113 (2002)], and so far only a few research groups have access to these tips. Therefore, it is necessary to develop more practical and economical methods for preparation of SMATs complementing the unique capabilities of nanotube tips, such as high resolution in imaging and control of tip orientation, for applications of AFM in a wide range of problems.
Silicon Surface Modification
Modification of silicon surfaces with a stable, uniform and ultrathin layer of biocompatible materials is of tremendous interest for the development of silicon-based bio-devices, including biochips, biosensors, microarrays, microfluidic systems, and implantable microdevices [J. Yakovleva, R. Davidsson, A. Lobanova, M. Bengtsson, S. Eremin, T. Laurell, J. Emneux, Anal. Chem. 2002,74, 2994; L. Leoni, D. Attiah, T. A. Desai, Sensors, 2002, 2, 111; S. Sharma, R. W. Johnson, T. A. Desai, Appl. Surf Sci. 2003, 206, 218]. Grafting oligo- or poly(ethylene glycol)s (OEGs or PEGs)—the well-known biocompatible materials—onto silicon oxide surfaces has been mostly based on siloxane chemistry using trichloro- or trialkoxylsilane derivatives [L. Leoni, D. Attiah, T. A. Desai, Sensors, 2002, 2, 111; S. Sharma, R. W. Johnson, T. A. Desai, Appl. Surf Sci. 2003, 206, 218; S.-W. Lee, P. E. Laibinis, Biomaterials 1998, 19, 1669; A. Papra, N. Gadegaard, N. B. Larsen, Langmuir 2001, 17, 1457]. Unfortunately, these reagents easily polymerize to form large aggregates and multilayers on the surfaces, and this is problematic particularly for coating on miniature devices. One way to circumvent this problem is to graft OH-terminated PEG onto Cl—Si surfaces prepared by chlorination of hydrogen-terminated silicon surfaces [X.-Y. Zhu, D. R. Staarup, R. C. Major, S. Danielson, V. Boiadjiev, W. L. Gladfelter, B. C. Bunker, A. Guo, Langmuir 2001, 17, 7798]. The above methods involve the formation of Si—O bonds with the surfaces. A more practical approach was envisioned based on hydrosilylation [M. R. Linford, P. Fenter, P. M. Eisenberger, C. E. D. Chidsey, J. Am. Chem. Soc. 1995, 117, 3145; R. L. Cicero, M. R. Linford, C. E. D. Chidsey, Langmuir 2000, 16, 5688; J. M. Buriak, Chem. Revs. 2002, 102, 1271; T. Strother, W. Cai, X. Zhao, R. J. Hamers, L. M. Smith, J. Am. Chem. Soc. 2000, 122, 1205; M. P. Steward, J. M. Buriak, Angew. Chem. Int. Ed. 1998, 37, 3257; Angew. Chem. 1998, 110, 3447; A. B. Sieval, R. Linke, G. Heij, G. Meijer, H. Zuilhof, E. J. R. Sudhölter, Langmuir 2001, 17, 7554; D. A. Nivens, D. W. Conrad, Langmuir 2002, 18, 499; R. Boukherroub, D. D. M. Wayner, J. Am. Chem. Soc. 1999, 121, 11513; A. B. Sieval, V. Vleeming, H. Zuilhof, E. J. R. Sudhölter, Langmuir 1999, 15, 8288] of α-OEG-ω-alkenes directly onto H-terminated silicon surfaces, forming Si—C bonds that are more stable towards hydrolysis than Si—O bonds. Also, the reaction can be induced by light, allowing for photopatterning the surface [M. P. Steward, J. M. Buriak, Angew. Chem. Int. Ed. 1998, 37, 3257; Angew. Chem. 1998, 110, 3447]. This very useful approach has not been reported, although hydrosilylation is widely used to prepare alkyl monolayers presenting a variety of surface functional groups including esters and amides [J. M. Buriak, Chem. Revs. 2002, 102, 1271]. An uncertainty was the presence of multiple ethylene glycol units that might interfere with the reaction and trap trace amount of water that facilitates the oxidation of the H—Si surface. While the initial interest of this approach was to modify silicon atomic force microscopic (AFM) tips [C. M. Yam, Z. Xiao, J. Gu, S. Boutet, C. Cai, J. Am. Chem. Soc. 2003, 125, 7498], this approach was also explored for the growth of OEG layers on other silicon surfaces. The research resulted in the following invention that can be used in silicon-based biotechnology. The following invention as described herein details the method of growth of OEG layers by hydrosilylation of CH2═CH(CH2)m1(CF2)m2(OCH2CH2)n OR where m1>0, m2≧0, n≧3, and R=alkyl groups such as CH3, amide, ester, thiolate, disulfide, or protected amino, hydroxy, or thiol groups, on hydrogen-terminated silicon surfaces including hydrogen-terminated flat silicon surfaces, porous silicon surfaces, and silicon nanoparticles. For example, m1=9, m2=0, n=3, 6, 7, 9, R=CH3, abbreviated as EG3 [S.-W. Lee, P. E. Laibinis, Biomaterials 1998, 19, 1669], EG6 [C. P. Fischer, C. Schmidt, H. Finkelmann, Macromol. Rapid Commun. 1995, 16, 435], and EG9 on atomically flat H—Si(111) surfaces (see FIG. 18).